Liposomal rehydration salt formulation and associated methods of use

ABSTRACT

A liposomal rehydration salt formulation used for preventing severe dehydration, maintaining body electrolytes and fluids in a human, and rehydrating a human includes phospholipids at a concentration of about 1.0 g/L to 10.0 g/L, salts, water, and a percentage inclusion ratio of salts (salts retained within total salts/liposomes) of at least 50% and a sodium electrolyte of about 12 mEq/L to 90 mEq/L, wherein said formulation has an actual osmolarity lower than 130 based on the at least 50% encapsulation of the salts and said liposomes comprise a particle diameter ranging from 200 nm to 500 nm.

PRIORITY APPLICATION(S)

This is a continuation-in-part application based on U.S. patentapplication Ser. No. 15/111,485 filed Jul. 14, 2016, which is based upona U.S. national stage application as international Application No.PCT/ES2015/070003 filed Jan. 7, 2015, which claims priority fromArgentina Patent Application No. P20140100123 filed Jan. 14, 2014, thedisclosures which are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to the technological field of improvedoral rehydration salts. In particular, it relates to liposomal oralrehydration salts.

STATE OF THE ART

References to oral rehydration salts in the form of liposomes are notabundant in literature. Several attempts to develop isolated products ofthis kind have been disclosed, which have not been successful.

It should be noted that U.S. Patent Publication No. 2005/0008685 (nowabandoned) describes the use of liposomes for preparing oral rehydrationsalts. However, the percentage inclusion ratio of salts (salts retainedwithin said liposomes/total salts) disclosed is 25%, which in spite ofimproving mouthfeel, still causes rejection by consumers or patients.

On the other hand, there are several reports on the benefits fromadministering liposomal rehydration salts, such as in “Absorption ofWater and Electrolytes from a Liposomal Oral Rehydration Solution: An invivo Perfusion Study of Rat Small Intestine” by P. K. Bardhan, A. S. M.Hamidur Rahman, Rifaat, and D. A. Sack—ICDDR,B: Centre for Health andPopulation Research, GPO Box 128, Dhaka 1000, Bangladesh, published inDecember 2003. This document makes reference to the improved mouthfeeland improved absorption mechanism of rehydration salts due to thepresence of liposomes.

Salt concentration as recommended by the WHO for rehydration salts isthe following:

ORS Concentration mmol/L Function Component g/L Glucose Na+ K+ Cl− Cit3−Rehydration Sodium 2.6 44.5 44.5 salts chloride Potassium 1.5 20.1Chloride Sodium 2.9 29.6 9.9 citrate Sweetener Glucose 13.5 74.9

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows TEM (Transmission Electron Microscopy) images of a liposomesample of the present invention after the final stage of the preparationprocess.

FIG. 2 illustrates the diameter distribution of the liposomes of thepresent invention formulation, wherein the particle size distribution ina DLS (Dynamic Light Scattering) analysis is shown.

FIG. 3 illustrates a calibration curve for turbidity measurement.

FIG. 4 represents the evolution of body mass in male animals accordingto Example 9.

FIG. 5 represents the evolution of body mass in female animals accordingto Example 9.

FIG. 6 represents the evolution of hematocrit concentration in maleanimals according to Example 9.

FIG. 7 represents the evolution of hematocrit concentration in femaleanimals according to Example 9.

FIG. 8 represents the evolution of sodium concentration (Natremia)(mmol/L) in male animals according to Example 9.

FIG. 9 represents the evolution of sodium concentration (Natremia)(mmol/L) in female animals according to Example 9.

FIG. 10 represents the evolution of potassium concentration (Kalemia)(mmol/L) in male animals according to Example 9.

FIG. 11 represents the evolution of potassium concentration (Kalemia)(mmol/L) in female animals according to Example 9.

SUMMARY OF THE INVENTION

A liposomal rehydration salt formulation comprises phospholipids at aconcentration of about 1.0 g/L to 10.0 g/L, salts, water, and apercentage inclusion ratio of salts (salts retained within totalsalts/liposomes) of at least 50% and a sodium electrolyte of about 12mEq/L to 90 mEq/L, wherein the formulation has an actual osmolaritylower than 130 based on the at least 50% encapsulation of the salts andthe liposomes comprise a particle diameter ranging from 200 nm to 500nm. The sodium electrolyte may be from about 35 mEq/L to 55 mEq/L. Theliposomal rehydration salt formulation may further comprise about 15mEq/L to 25 mEq/L of potassium electrolyte. The phospholipids may beselected from the group consisting of phosphatidylcholines (PCs),phosphatidylserines (PSs), phosphatidylethanolamines (PEs),phosphatidylglycerols (PGs), phosphatidylinositols (PIs), phosphatidicacids (PAs), and mixtures thereof. The composition may further comprisesan antioxidant selected from the group consisting of phytosterol,tocopherol, and mixtures thereof.

The salts may be selected from the group consisting of sodium chlorideat a concentration of 0.7 g/L to 2.8 g/L, potassium citrate at aconcentration of 0.8 g/L to 2.5 g/L, sodium citrate at a concentrationof 0.5 g/L to 2.9 g/L, and mixtures thereof. The formulation may furthercomprise about 10 g/L to 17 g/L of glucose and about 8.0 g/L to 15 g/Lof at least one additional sugar. The formulation may further compriseStevia at a concentration of about 0.1 g/L to 0.25 g/L. The formulationmay further comprise natural flavours at a concentration of about 1 g/Lto 3.5 g/L.

A method of preventing severe dehydration and maintaining bodyelectrolytes and fluids in a human comprises orally administering aliposomal rehydration salt formulation comprising phospholipids at aconcentration of about 1.0 g/L to 10.0 g/L, salts, water, and apercentage inclusion ratio of salts (salts retained within totalsalts/liposomes) of at least 50% and a sodium electrolyte of about 12mEq/L to 90 mEq/L, wherein the formulation has an actual osmolaritylower than 130 based on the at least 50% encapsulation of the salts andthe liposomes comprise a particle diameter ranging from 200 nm to 500nm. The rehydration salt formulation may be formulated for oraladministration for use by humans that are pregnant or breast-feeding orengaged in one or more of sport exercises, outdoor activities, extremeweather activities, climbing and flying. The liposomal rehydration saltformulation may also be formulated for oral administration for use bypatients having one or more of stomach ailments, skin burns, parenteralor enteral nutrition ailments, celiac disorders, diabetes, SGLT2inhibitor treatment disorders, intestinal failure, Short Bowel Syndrome,Cycling Vomiting Syndrome, Gastroparesis, Postural OrthostaticTachycardia Syndrome, Ulcerative Colitis, Colon Cancer, Dysphagia,Sjogren Syndrome, Crohn's disease, Lupus, Alzheimer's disease, Renalcomplications, HIV, Inflammatory Bowel Disease, an Ostomy, MicrovillusInclusion Disease, and Cystic Fibrosis.

The sodium electrolyte may be from about 35 mEq/L to 55 mEq/L. Theliposomal rehydration salt formulation may include a potassiumelectrolyte and administering about 15 mEq/L to 25 mEq/L of thepotassium electrolyte. The liposomal rehydration salt formulation maycomprise about 10 g/L to 17 g/L of glucose and 8.0 g/L to 15 g/L of atleast one additional sugar.

A method of rehydrating a human suffering from dehydration comprisesorally administering a liposomal rehydration salt formulation comprisingphospholipids at a concentration of about 1.0 g/L to 10.0 g/L, salts,water, and a percentage inclusion ratio of salts (salts retained withintotal salts/liposomes) of at least 50% and a sodium electrolyte of about12 mEq/L to 90 mEq/L, wherein the formulation has an actual osmolaritylower than 130 based on the at least 50% encapsulation of the salts andthe liposomes comprise a particle diameter ranging from 200 nm to 500nm.

DETAILED DESCRIPTION

The liposomal rehydration salt formulation of the present inventioncontains phospholipid liposomes, preferably selected from the groupconsisting of phosphatidylcholines (PCs), phosphatidylserines (PSs),phosphatidylethanolamines (PEs), phosphatidylglycerols (PGs),phosphatidylinositols (PIs), phosphatidic acids (PAs), and mixturesthereof, at a concentration of less than 6% (W/V); and optionally anantioxidant selected from phytosterol, tocopherol, and mixtures thereof,at a concentration of 0.2 to 0.5% (W/V); water; salts selected from thegroup consisting of sodium chloride at a concentration of 0.7 to 2.8g/l, potassium citrate at a concentration of 0.8 to 2.5 g/l, sodiumcitrate at a concentration of 0.5 to 2.9 g/l, and mixtures thereof;optionally, it may further comprise carbohydrates, among which glucoseis preferred.

Intestinal salt absorption mechanisms are enterocyte co-transportsystems. These systems involve carrying salts into the body along withother molecules, glucose being the most important among them. This iswhy rehydration salt formulations targeting hyponatremia, associatedboth with sports and acute diarrhea, are composed of a mixture of saltsand glucose. Salt concentration should be higher than that of the body,so that glucose-mediated transport can be enabled by an osmotic gradientallowing for incorporation of salts through membranes. However, glucoseintake is restricted by the calorie intake of this molecule.

Liposomes are nanoparticles consisting of a phospholipid bilayer, thesame as cell membranes of enterocytes. Based on different mechanisms,liposomes (and all the contents carried in them) are highly capable ofbeing absorbed by the small intestine cells, increasing bioavailabilityof the transported actives. Liposomal rehydration salt formulations aimat providing transport mechanisms of liposomes to the basic mechanismsof salts. In vivo tests have shown that an encapsulated ORS formulationhaving salt concentrations in accordance with WHO standards causes a1.39-fold hydration increase in animals under normal conditions, ascompared to the WHO recommended formula, and a 1.45-fold hydrationincrease in animals infected with cholera as compared to the WHOrecommended formula (“Absorption of Water From a Liposomal OralRehydration Solution: an In Vivo Perfusion Study of Rat Small IntestineExposed to Cholera Toxin” Gastroenterology—Volume 142, Issue 5,Supplement 1, Pages S-21, May 2012—Pradip K. Bardhan, Nasirul Islam,Rifat Faruqui).

In view of the above, one of the great advantages of the presentinvention relies on the use of lower carbohydrate concentrations,ranging from 0 to 6 g/l, which improves mouthfeel and tolerance to theformulation. Furthermore, it would be possible to replace glucose with amixture of carbohydrates such as fructose, dextrose, high fructose cornsyrup and mixtures thereof, and even with artificial sweeteners such assucralose. Low glucose concentration is very important in sport drinks.It is even possible to accomplish efficient rehydration in the absenceof glucose, which would allow the formulation to be consumed bydiabetics.

In addition, and also due to lower glucose concentration, theformulation of the present invention exhibits reduced osmolality withrespect to commercially-available formulations, also lower than 190mmol/L, which accounts for the possibility of accomplishing efficientrehydration without running the risk of inducing hypernatremia in thepatient.

Furthermore, one of the novel aspects of this invention is the fact thatit significantly improves percentage inclusion ratio of salts (saltsretained within said liposomes/total salts) with respect to the priorart. This ratio is at least 40%, preferably at least 50%, morepreferably at least 52%. In a preferred alternative of the presentinvention, said percentage inclusion ratio is at least 56%. Theseinclusion ratio values have not been previously disclosed in the priorart, and they allow for the preparation of formulations containing lowersalt concentrations with improved rehydration effects, as disclosed inthe present invention. This inclusion ratio is achieved by usingtangential ultrafiltration method. Although well-known, this method hasnever been employed to increase the ratio of oral rehydration saltsencapsulated within liposomes to the total amount of the salts of theformulation, thereby solving the technical problem of rejection causedby oral rehydration salts due to their unpleasant taste.

It has been demonstrated in Example 4 that encapsulation of more than50% of the salts in the formulation of the present invention causesunpleasant taste inherent to salts to be almost imperceptible. Thisfacilitates consumption by children younger than 12 years, who representthe most affected population in terms of acute dehydration.

Furthermore, the liposomes of the formulation of the invention areproduced such that the particle diameter ranges from 200 to 500 nm;preferably from 225 to 450 nm, as shown in FIG. 2.

The liposomal rehydration salt formulation of the present invention isan oral administration infusion for oral replacement of fluids andelectrolyte salts in the treatment of dehydration caused by diarrhea andvomiting, prevention of severe dehydration, and maintenance of bodyelectrolytes and liquids. The present invention may also be an oraladministration infusion for use in sport activities.

The process for preparing the formulation of the invention comprises thefollowing steps:

a. preparing an aqueous phase (AP) or buffer comprising sodium chloride,potassium citrate, sodium citrate dissolved in distilled water;

b. separately preparing an ethanol phase (EP), by dissolving saidphospholipid at a concentration of 0.1 to 6% (W/V), and optionally anantioxidant at a concentration of 0.2 to 0.5% (W/V) in alcohol,preferably ethyl alcohol;

c. inducing formation of liposomes by injecting said EP into said AP atroom temperature, while stirring;

d. subjecting the liposomal solution obtained in step “c” to atangential ultrafiltration (TUF) concentration process, removing thebuffer and maintaining the liposomes and their contents, thus reducingthe volume at least by 10-fold;

e. subjecting the liposomal solution obtained in step “d” to atangential ultrafiltration (TUF) concentration process, wherein ethanolis eliminated and the buffer is replaced with saline solution, andmaintaining the liposomes and their contents.

In step “a”, said aqueous phase (AP) or buffer comprises sodium chlorideat a concentration of 6 to 20 mmol/l, potassium citrate at aconcentration of 1 to 12 mmol/l, sodium citrate at a concentration of 2to 5 mmol/l, and distilled water.

In step “e” of the process of the present invention, said salinesolution comprises a sodium concentration of 12 to 50 mmol/l, apotassium concentration of 3 to 36 mmol/l, a chloride concentration of15 to 40 mmol/l, a citrate concentration of 8 to 17 mmol/l, and itfurther comprises glucose at a concentration of 17 to 45 mmol/l.

Furthermore, the AP:EP volume ratio in step “c” is at least 10:1;preferably at least 10:0.5; more preferably at least 10:0.4.

The process of the present invention comprises a perpendicular flowprocess, wherein the ethanol phase is added on the aqueous phase byperpendicular coupling to the flow of the former, and with a linearvelocity ratio REP/RAP of no more than 1/200.

EXAMPLES Example 1

Preparation of the Liposomal Rehydration Salt Formulation of theInvention

a) Preparation of the Ethanol Phase (EP)

25 g of purified soybean phosphatidylcholine and 0.5 L of ethanol areadded, heated to 65° C., and stirred until completely dissolved. A totalamount of 2.5 g of mixed tocopherols (ascorbyl palmitate andD-Alpha-Tocopherol) is added as antioxidant. The solution is left torest until it reaches room temperature.

b) Preparation of the Saline Aqueous Phase (AP)

4.33 g Sodium chloride, 3.42 g Potassium citrate, and 4.83 g Sodiumcitrate are dissolved in 4.5 L water and stirred at room temperatureuntil completely dissolved.

c) Production of Liposomes

0.5 L of Ethanol phase is slowly added on 4.5 L of Aqueous phase undercontinuous circular stirring. This may be also performed by means of aCross-Flow or Perpendicular Flow process, wherein the Ethanol phase isadded on the Aqueous phase by perpendicular coupling to the flow of theformer, and with a linear velocity ratio, REP/RAP, of no more than1/200. FIG. 1 shows liposomes formed with both processes. FIG. 2 showsthe results of particle size distribution in DLS (Dynamic LightScattering) analysis.

d) Increasing Encapsulation Efficiency

Ultrafiltration without recirculation, by tangential flow, is carriedout so as to remove the aqueous phase solutes that are not trapped inthe liposomes. This process is completed after removing 90% volume ofthe previous liposomal dispersion.

e) Buffer substitution

Ultrafiltration by tangential flow is carried out to remove ethanol fromthe liposomal salt solution. While the process is conducted, thesolution is fed at a speed equal to the permeation speed with an aqueoussolution of Sodium chloride (1.05 mg/ml), Potassium citrate (0.83mg/ml), Sodium citrate (1.17 mg/ml) and Glucose (6.75 mg/ml).

The liposomal rehydration salt formulation of the present invention isthereby obtained, said formulation having the following features:

-   -   Percentage inclusion ratio of salts (salts retained within        liposomes/total salts) of 56.48%    -   Chloride concentration: 39.7 mmol/L    -   Citrate concentration: 16 mmol/L    -   Potassium concentration: 17.9 mmol/L    -   Sodium concentration: 69.7 mmol/L    -   Glucose concentration: 33.0 mmol/L

Example 2

Process for Preparing the Present Invention Formulation with aPercentage Inclusion Ratio of Salts of 56%

Stage a

A solution of 4.5 L distilled water with salts is prepared at thefollowing concentration:

Concentration (mmol/L) Glucose Na K Cl Cit Sodium chloride 14.82 14.82Potassium citrate 6.70 2.23 Sodium citrate 11.23 3.74 Glucose —

Stage b

Separately, a solution of Phosphatidylcholine in 500 ml of 5% ethylalcohol (W/V) is prepared.

Stage c

Formation of liposomes is induced by injecting the ethanol solution intothe aqueous phase while stirring. Then 15% of the salts areencapsulated; therefore, internal and external salt concentrations areas follows:

Internal External Na 3.91 22.14 K 1.00 5.70 Cl 2.23 12.60 Cit 0.8955.074 Glucose 0 0

Stage d

Five (5) liters of liposomal ORSs are subjected to a tangentialultrafiltration concentration process. This process allows for removingthe buffer without eliminating the liposomes and their contents. Thisprocess is performed until the volume is reduced by 10-fold. At the endof the process, 500 ml of liposomal salts having the followingconcentration is obtained.

Internal External Na 39.1 22.14 K 10.0 5.70 Cl 22.3 12.60 Cit 8.95 5.074Glucose 0 0

Stage e

At this stage, the buffer is substituted by using the TUF process again.In this case, the total volume is reduced by 10-fold, and replaced withan aqueous solution with the following salt concentration.

Concentration (mmol/L) Na 31.57 K 8.13 Cl 17.96 Cit 7.24 Glucose 40.70

Accordingly, 500 ml of a solution of liposomal ORSs having the followingsalt concentration is obtained.

Internal External TOTAL Na 39.1 30.64 69.74 K 10.0 7.88 17.88 Cl 22.317.45 39.75 Cit 8.95 7.02 15.97 Glucose 0 33.03 33.03

The so obtained formulation exhibits a salt concentration equal to thatof the formulation recommended by the WHO, with an encapsulationefficiency of about 56.05%. Other features recommended by the WHO andUNICEF in their joint statement issued in May 2004 and accomplished inthis invention are reduced glucose content and lower osmolality.

The liposomal rehydration salt formulation of the present invention isthereby obtained, said formulation having the following features:

-   -   Percentage inclusion ratio of salts (salts retained within        liposomes/total salts) of 56.05%    -   Chloride Concentration: 39.75 mmol/l    -   Citrate Concentration: 15.97 mmol/l    -   Potassium Concentration: 17.88 mmol/l    -   Sodium Concentration: 69.74 mmol/l    -   Glucose Concentration: 33.03 mmol/l

Example 3

Encapsulation Efficiency Using the Barium Sulphate Turbidity Method

Two phosphatidylcholine ethanol solutions are prepared, one of themnamed “FE1”, which has a concentration of 2% Phosphatidylcholine (thesame as the one used in the TLEC formulation of U.S. Patent PublicationNo. 2005/0008685), and the other named “FE2”, with a concentration of 5%Phosphatidylcholine (the same as the one used in the present invention).

Separately, an aqueous solution (AP) of 56.23 mM ammonium sulphate isprepared (this concentration reproduces the ionic strength of the WHOrehydration salts).

Two liposomal solutions are then prepared using the ethanol phaseinjection method, in which 10 ml FE1 and FE2 are separately injected intwo fractions of 90 ml AP under magnetic stirring at 300 rpm and 25° C.Consequently, 100 ml of two liposomal formulations are obtained:

-   -   *1:10 (v/v) FE1:AP (LIPO-1)    -   *1:10 (v/v) FE2:AP (LIPO-2)

The LIPO-2 formulation was subjected to a tangential ultrafiltrationprocess using a hollow fiber cartridge with a 300 KD cut off, withoutfeedback. Ultrafiltration continued until reducing the volume by10-fold. This is the process we carry out in our invention in order toobtain higher encapsulation efficiency.

Samples are taken from both final solutions.

Then, 10 ml of each formulation (LIPO-1 and LIPO-2) is taken andultrafiltered by using the same system but feeding back each formulationwith 126 mM sucrose aqueous buffer. Thus, the sulphate ionsnon-encapsulated into liposomes are eliminated from each solution andsubstituted with a solution having the same osmolality in order toensure integrity of the liposomal membranes.

Then 5 ml of each formulation is taken before and after theultrafiltration process, and 10% surfactant Triton X-100 is added toeach of them, in order to break the lipid membranes. This solution iskept under stirring at 25° C. for 1 hour.

Turbidity Measurement

Soluble sulphates precipitate in the presence of barium chloride in theform of barium sulphate (BaSO4) as a white solid. Measurement of tancereduction as a consequence of the presence of barium sulphate, to acertain wave length in a UV/Vis spectrometer allows for determination ofthe sulphate ion concentration in aqueous solution.

For experimental determination, a calibration curve was plotted by using50 ml ammonium sulphate patterns with the following concentrations: 0.1mM, 0.25 mM, 0.5 mM, 0.75 mM and 1 mM. An excess of 31.23 mg (3 mM)barium chloride was added to each solution. Then, transmittance of eachsolution was determined in triplicate by using an UV/VIS spectrometer(Jenway 7315). The calibration curve is shown in FIG. 3.

Then, 0.1 ml of the unknown LIPO-1 solution before ultrafiltration and 1ml of the solution after ultrafiltration were taken, both alreadytreated with surfactant, and the same excess of barium chloride wasadded (3 mM). Transmittance of the sample before ultrafiltration was49.22%±0.17%, corresponding to [Sulphate]=0.5273 mM. Taking dilutionsinto account, the total sulphate concentration before ultrafiltration is52.73 mM. Transmittance of the sample after ultrafiltration was48.25%±0.32%, corresponding to [Sulphate]=0.5091 mM. Taking dilutionsinto account, the salt concentration after ultrafiltration is 5.091 mM.This indicates that the ratio of sulphate encapsulated in theformulation produced with the concentration of Phosphatidylcholine ofU.S. Patent Publication No. 2005/0008685 was 10.59%.

Likewise, 0.1 ml of the unknown LIPO-2 solution before ultrafiltration,and 0.1 ml of the solution after ultrafiltration were taken, bothalready treated with surfactant, and the same excess of barium chloridewas added (3 mM). Transmittance of the sample before ultrafiltration was73.19%±0.19%, corresponding to [Sulphate]=0.9818 mM. Taking dilutionsinto account, the total sulphate concentration before ultrafiltration is98.18 mM. Transmittance of the sample after ultrafiltration was50.66%±0.24%, corresponding to [Sulphate]=0.5545 mM. Taking dilutionsinto account, the salt concentration after ultrafiltration is 55.45 mM.This indicates that the ratio of sulphate encapsulated in theformulation of the present invention was 56.48%.

Note: The results are directly proportional to the encapsulatedrehydration salts, since all the salts have similar and increased watersolubility. Therefore, the encapsulation level by the method of liposomeformation by ethanol phase injection is statistical, and it will besimilar in compounds with similar water solubility.

Example 4

Multicenter, Randomized and Single-Blind Mouthfeel Assay

Liposomal Rehydration Salts Samples:

Formula A: Liposomal rehydration salt formulation of example 3 of thepresent invention.

Formula B: Liposomal rehydration salts according to example 3 of U.S.Patent Publication No. 2005/0008685 A1.

Methodology

Healthy individuals from 21 to 40 years of age were recruited. Thoseindividuals with cardiac or renal diseases, diabetics, individuals whohad suffered from diarrhea the month prior to the assay, individualsaffected by rhinitis, or individuals under antibiotic or iron treatmentwere excluded from the assay.

The screening of the individuals took place in four different shoppingmalls in the city of Santa Fe, Argentina. After explaining the test tothe individuals and having them signed their consent (either bythemselves or by their parents or legal guardians in case of underagepeople), the individuals were randomized. Randomization indicates theorder in which the two formulations would be tasted. In order to getfamiliar with this type of flavors, the individuals took a little sip ofthe two formulations and then rinsed their mouths with water and a pieceof salt-free bread. Thereafter, they tasted the two formulations in theorder indicated by randomization, and they were asked to indicate theformulation of their preference. The same test was repeated twice withboth formulations, after a new mouth rinse with water and pieces ofbread. They were offered each formulation in amounts of less than 20 mlin total, inside red plastic glasses (to avoid color influence on thedecision). The formulations were administered at room temperature,without any refrigeration.

Each individual tasted both formulations repeatedly (twice the firsttasting and twice the second tasting); to corroborate consistency bothtimes each tasting took place, kappa(k) statistic was used (URL:www.graphpad.com/quickcalcs/kappa2.cfm) as well as a 95% CI.

Results

120 individual were studied, out of which 4 individuals did not meet theinclusion criteria (out of age), so the final test cohort consisted of116 individuals with an average of 30-32 years old. The distribution ofthe individuals per shopping mall was similar: between 27 and 30 pereach shopping mall. 59 individuals were female (50.9%).

Regarding the results obtained, we found very high consistency betweenthe scores of the 2 tests with the same formulations, both in the firsttasting (k=0.91; 95% CI: 0.85-0.98), and the second tasting (k=0.87; 95%CI: 0.80-0.94). Therefore, in the statistical analysis, it was decidedto use the results corresponding to the second time each of the twotastings was scored.

Out of the 116 individuals, 97 individuals preferred the taste offormula A, 2 preferred the taste of formula B. 17 individuals were notcertain as to which they preferred, so they were not counted.

Example 5

Process for Preparing the Formulation of the Present Invention with aPercentage Inclusion Ratio of Salts of 56% (for Sport Activities)

Stage a

A solution of 4.5 L distilled water is prepared with salts at thefollowing concentration:

Concentration (mmol/L) Glucose Na K Cl Cit Sodium chloride 6.01 6.01Potassium citrate 3.86 1.29 Sodium citrate 6.02 2.01 Glucose —

Stage b

On the other hand, a solution of phosphatidylethanolamine in 500 ml of4% Ethyl Alcohol (W/V) is prepared.

Stage c

Liposome formation is induced by injecting the ethanol solution into theaqueous phase while stirring. Here, 15% of the salts are encapsulated.Therefore, the internal and external salt concentrations are thefollowing:

Internal External Na 1.57 10.46 K 0.50 3.36 Cl 0.78 5.23 Cit 0.49 2.81Glucose 0 0

Stage d

The Five (5) liters of liposomal ORSs are subjected to a tangentialultrafiltration (TUF) concentration process. This process allows forremoving the buffer without eliminating the liposomes and theircontents. This process is carried out until reducing the volume by10-fold. At the end of the process, 500 ml liposomal salts having thefollowing concentration are obtained.

Internal External Na 15.7 10.56 K 5.04 3.46 Cl 7.84 5.23 Cit 4.90 2.81Glucose 0 0

Stage e

At this stage, buffer substitution is performed, again with the TUFprocess. In this case, the total volume is reduced by 10-fold andreplaced with an aqueous solution having the following saltconcentration:

Concentration (mmol/L) Na 12.56 K 3.65 Cl 5.65 Cit 3.04 Glucose 17.80

This buffer further contains Stevia (Reb A 97—PureCircle) at aconcentration of 0.15 g/L; Sucrose at a concentration of 28.5 g/L;Citric Acid at a concentration of 3.6 g/L; and Natural Flavors at aconcentration of 1.5 g/L.

Accordingly, 500 ml of a liposomal ORS solution is obtained, containing40 g/l phospholipid, with the following salt concentration:

Internal External TOTAL Na 15.7 12.35 28.05 K 5.04 3.62 8.66 Cl 7.845.61 13.45 Cit 4.90 3.02 7.92 Glucose 0 16.02 16.02

The formulation of the present example is useful for people in need ofhydration due to sun exposure, illness, pregnancy, travel fatigue,hangover, mental stress, strenuous work, or just living an active life.It may be produced with orange, strawberry, apple, pear, blueberry,raspberry flavors, among others.

Example 6

Process for Preparing the Formulation of the Present Invention with aPercentage Inclusion Ratio of Salts of 56%

Pediatric Rehydration Formulation. Stage a

A solution of 4.5 L distilled water is prepared with salts at thefollowing concentration:

Concentration (mmol/L) Glucose Na K Cl Cit Sodium chloride 14.82 14.82Potassium citrate 6.70 2.23 Sodium citrate 11.23 3.74 Glucose —

Stage b

On the other hand, a solution of phosphatidylserine in 500 ml of 3%Ethyl alcohol (W/V) is prepared.

Stage c

Liposome formation is induced by injecting the ethanol solution into theaqueous phase while stirring. Here, 15% of the salts are encapsulated.Therefore, the internal and external salt concentrations are thefollowing:

Internal External Na 3.91 22.14 K 1.00 5.70 Cl 2.23 12.60 Cit 0.8955.074 Glucose 0 0

Stage d

The Five (5) liters of Liposomal ORSs are subjected to a tangentialultrafiltration concentration process. This process allows for removingthe buffer without eliminating the liposomes and their contents. Thisprocess is carried out until the volume is reduced by 10-fold. At theend of the process 500 ml of liposomal salts having the followingconcentration is obtained.

Internal External Na 39.1 22.14 K 10.0 5.70 Cl 22.3 12.60 Cit 8.95 5.074Glucose 0 0

Stage e

At this stage, buffer substitution is carried out, again with the TUFprocess. In this case, the total volume is reduced by 10-fold andreplaced with an aqueous solution having the following saltconcentration:

Concentration (mmol/L) Na 31.57 K 8.13 Cl 17.96 Cit 7.24 Glucose 40.70

This buffer further contains Sucralose at a concentration of 0.12 g/L;high fructose corn syrup (55° Brix) at a concentration of 33.3 g/L;Citric Acid at a concentration of 4.0 g/L; and Natural Flavors at aconcentration of 1.7 g/L.

Accordingly, 500 ml of a liposomal ORS solution with 30 g/lphosphatidylserine and the following salt concentration is obtained:

Internal External TOTAL Na 39.1 30.64 69.74 K 10.0 7.88 17.88 Cl 22.317.45 39.75 Cit 8.95 7.02 15.97 Glucose 0 33.03 33.03

The formulation of the present example is useful for children sufferingfrom vomiting or diarrhea under the risk of dehydration, and it may beproduced with orange, strawberry, apple, pear, blueberry, raspberryflavors, among others.

Example 7

Process for Preparing the Formulation of the Present Invention with aPercentage Inclusion Ratio of Salts of 56%

Stage a

A solution of 4.5 L distilled water is prepared with salts at thefollowing concentration:

Concentration (mmol/L) Glucose Na K Cl Cit Sodium chloride 6.01 6.01Potassium citrate 3.86 1.29 Sodium citrate 6.02 2.01 Glucose —

Stage b

On the other hand, a solution of Phosphatidylcholine in 500 ml of 5%Ethyl alcohol (W/V) is prepared.

Stage c

Liposome formation is induced by injecting the ethanol solution into theaqueous phase while stirring. Here, 15% of the salts are encapsulated.Therefore, the internal and external salt concentrations are thefollowing:

Internal External Na 1.57 10.46 K 0.50 3.36 Cl 0.78 5.23 Cit 0.49 2.81Glucose 0 0

Stage d

The five (5) liters of Liposomal ORSs are subjected to a tangentialultrafiltration concentration process. This process allows for removingthe buffer without eliminating the liposomes and their contents. Thisprocess is carried out until the volume is reduced by 10-fold. At theend of the process, 500 ml of liposomal salts having the followingconcentration is obtained.

Internal External Na 15.7 10.46 K 5.04 3.46 Cl 7.84 5.23 Cit 4.90 2.81Glucose 0 0

Stage e

At this stage, buffer substitution is carried out, again with the TUFprocess. In this case, the total volume is reduced by 10-fold andreplaced with an aqueous solution having the following saltconcentration:

Concentration (mmol/L) Na 12.56 K 3.65 Cl 5.65 Cit 3.04 Glucose 17.80

This buffer further contains Stevia (Reb A 97—PureCircle) at aconcentration of 0.13 g/L; Sucrose at a concentration of 22.2 g/L;Citric Acid at a concentration of 3.4 g/L; and Natural Flavors at aconcentration of 1.5 g/L.

Accordingly, 500 ml of a liposomal ORS solution is obtained having thefollowing salt concentration:

TOTAL Internal External (mmol/L) Na 15.7 12.35 28.05 K 5.04 3.62 8.66 Cl7.84 5.61 13.45 Cit 4.90 3.02 7.92 Glucose 0 16.02 16.02

This formulation may be suitable for consumption by sportspeople.

Example 8

Process for Preparing the Formulation of the Present Invention with aPercentage Inclusion Ratio of Salts of 56% for High-PerformanceSportspeople

Stage a

A solution of 4.5 L distilled water is prepared with salts at thefollowing concentration:

Concentration (mmol/L) Glucose Na K Cl Cit Sodium chloride 6.01 6.01Potassium citrate 3.86 1.29 Sodium citrate 6.02 2.01 Glucose —

Stage b

On the other hand, a solution of phosphatidylinositol in 500 ml of 5%Ethyl alcohol (W/V) is prepared.

Stage c

Liposome formation is induced by injecting the ethanol solution into theaqueous phase while stirring. Here, 15% of the salts are encapsulated.Therefore, the internal and external salt concentrations are thefollowing:

Internal External Na 1.57 10.46 K 0.50 3.36 Cl 0.78 5.23 Cit 0.49 2.81Glucose 0 0

Stage d

The 5 Liters of Liposomal ORSs are subjected to a (TUF) tangentialultrafiltration concentration process. This process allows for removingthe buffer without eliminating the liposomes and their contents. Thisprocess is carried out until the volume is reduced by 10-fold. At theend of the process, 500 ml of liposomal salts having the followingconcentration is obtained.

Internal External Na 15.7 10.46 K 5.04 3.46 Cl 7.84 5.23 Cit 4.90 2.81Glucose 0 0

Stage e

At this stage, buffer substitution is carried out, again with the TUFprocess. In this case, the total volume is reduced by 10-fold andreplaced with an aqueous solution having the following saltconcentration:

Concentration (mmol/L) Na 12.56 K 3.65 Cl 5.65 Cit 3.04 Glucose 0

This buffer further contains high fructose corn syrup (55° Brix) at aconcentration of 3.22 g/L; Vitamin B1 at a concentration of 0.002 g/L;Vitamin B5 at a concentration of 0.011 g/L; Vitamin B6 at aconcentration of 0.011 g/L; Citric Acid at a concentration of 3.6 g/L;and Natural Flavors at a concentration of 1.5 g/L.

Accordingly, 500 ml of a liposomal ORS solution with 50 g/lphosphatidylinositol and the following salt concentration is obtained:

Internal External TOTAL Na 15.7 12.35 28.05 K 5.04 3.62 8.66 Cl 7.845.61 13.45 Cit 4.90 3.02 7.92 Glucose 0 16.02 16.02

This formulation may be suitable for consumption by high-performancesportspeople.

Example 9

Preclinical Assay of the Rehydration Salt Formulation of the PresentInvention

A batch of pediatric liposomal rehydration salts of Example 6 of thepresent invention as a finished product is compared to commercialproduct Pedialyte (Abbott Laboratories) taken as reference substance.Said comparison encompassed the development of an osmotic diarrhea modelin rats for efficiency evaluation.

Experimental Design:

An osmotic diarrhea experimental model was developed as described inWapnir et al., 1988,1991 (Am.J.Clin. Nutr. 1988; 4784-90; J. Pediatr.1991; 118:S53-61). Four experimental animal groups were used, eachconsisting of 10 animals (5 male and 5 female animals). Groups 1, 2 and3 were induced diarrhea by replacing the water for an oral solution of50% magnesium citrate (USP XXII) for 5 days. Group 4 was not induceddiarrhea and was allowed to drink water during said period. Onceinduction was completed, Group 1 was treated with the test substance;Group 2 was treated with the reference substance; Group 3 receivedphysiological solution; while Group 4 was not treated at all. Bodyweight, Natremia, Kalemia, and Hematocrit variables were analyzed bothduring treatment and 12 hours after completion. Young female and maleWistar rats with genetic certification were used. They were divided intosubgroups, placed into jails, and identified with a correlative integernumber.

The animals were kept under controlled ambient conditions: temperaturebetween 22±3° C., controlled photoperiod (12 hs light/12 hs darkness)and free access to commercial food and water. MicroVENT rack systemsprovided by Allentown Inc., European Type IIIH (POE GC-065) models, wereemployed.

Forty (40) animals divided into four experimental groups (each groupcomprising 5 male and 5 female animals) were used.

Group 1: (5 male and 5 female animals). It was distributed into 2subgroups: 1-M; 1-F, each consisting of 5 animals of the same sex. Theseanimals were subjected to osmotic diarrhea induction and treated withthe test substances.

Group 2: (5 male and 5 female animals). It was distributed into 2subgroups: 2-M; 2-F, each consisting of 5 animals of the same sex. Theseanimals were subjected to osmotic diarrhea induction and treated withthe reference substance.

Group 3: (5 male and 5 female animals). It was distributed into 2subgroups: 3-M; 3-F, each consisting of 5 animals of the same sex. Theseanimals were subjected to osmotic diarrhea induction and treated withphysiological solution.

Group 4: (5 male and 5 female animals). It was distributed into 2subgroups: 4-M; 4-F, each consisting of 5 animals of the same sex. Theseanimals did not receive any treatment.

Treatment: Osmotic Diarrhea Induction:

In groups 1, 2, and 3, an osmotic diarrhea experimental model wasdeveloped as described in Wapnir et al., 1988,1991 (Am.J.Clin. Nutr.1988;4784-90; J. Pediatr. 1991;118:S53-61).

Test Substances:

Liposomal Rehydration Salts—Pediatric Formulation of Example 6 of thepresent invention.

Reference Substance:

Rehydration Salts Pedialyte—Pediatric Formulation manufactured by AbbottLaboratories.

Dosage and Administration:

Groups 1, 2, and 3 were orally administered a total dose of 125ml/kg/day of the different test substances, distributed in 12 doses at aone-hour-interval between each other. The dose was selected taking intoaccount the dosing instructions of Pedialyte according to which doses of100 to 150 ml/kg are recommended. The dosage volume of administrationwas calculated according to the average weight of the male and femalerats obtained on Day 5 during the morning, at the time magnesium citratesolution was removed and treatment was initiated, thereby determiningdifferential doses for male and female rats.

The volume corresponding to each dosage was calculated according to thefollowing formula:

${V\left( \frac{ml}{animal} \right)} = \frac{P \times 125}{12 \times 1000}$

Wherein P is the average weight in grams, either of the male or femalerats, as applicable.

Dose administration took place every hour beginning at 9 A.M. on Day 5.

Hematocrit Determination:

Upon the extraction of one drop of blood, a microhematocrit wasconducted by using heparinized microtubes. The samples were collected atthe following times:

-   -   Day 5: 08:00 hs, 12:00 hs, 16:00 hs and 20:00 hs.

The collected samples were also subjected to Natremia and Kalemiadetermination.

Data Analysis: A comparative analysis of the different formulations wasperformed through descriptive statistics and two-way analysis ofvariance (ANOVA), followed by Tukey's multiple comparison test toidentify differences between different times. These operations wereperformed with GraphPad Prism 6.0 software.

Results Body Mass Recovery:

Media SEM Media SEM Media SEM Media SEM 1 M 2 M 3 M 4 M Day 0 137.23.44093 137.8 2.332381 134.2 2.2 138.4 1.805547 Day 1 133.8 2.61534 1342.387467 130.6 2.357965 143.4 2.014944 Day 2 128.2 2.374868 128 1.760682123.8 3.168596 143.8 1.714643 Day 3 122.8 2.596151 124.4 3.091925 120.63.37046 146.2 2.853069 Day 4 112.8 4.465423 113.8 2.477902 110.6 3.17175149.6 2.088061 Day 5 (8 hours) 108.6 3.613863 105 2.258318 103.42.158703 153.4 3.249615 Day 5 (12 hours) 115.8 4.140048 110.4 1.939072109 2.50998 153.4 2.501999 Day 5 (16 hours) 124.8 4.476605 115.61.122497 114 2.097618 155.8 2.557342 Day 5 (20 hours) 133.4 4.905099121.4 0.6 119.6 2.249444 156.6 2.61916 Day 6 137.1 5.416641 125.41.939072 124.4 3.043025 157.8 2.416609 1 H 2 H 3 H 4 H Day 0 118.40.9273618 120.4 1.50333 120 0.9486833 119.4 2.521904 Day 1 112.61.536229 114.6 1.469694 115 1.264911 121.2 2.709244 Day 2 105.6 1.28841109.6 1.28841 107.8 2.034699 123.8 2.61534 Day 3 98.8 1.714643 1021.30384 102.8 1.907878 124.4 2.785677 Day 4 93.4 1.249 93.6 1.939072 960.8944272 126.8 2.2 Day 5 (8 hours) 92.8 1.939072 89.8 1.827567 93.22.437212 128.6 2.135416 Day 5 (12 hours) 106.4 1.6 103 2.213594 107.80.9695359 127 1.949359 Day 5 (16 hours) 112.6 1.4 101.6 1.860107 108.61.886796 126 2.073644 Day 5 (20 hours) 116.6 1.886796 102.6 1.469694113.2 1.157584 123.4 2.420743 Day 6 118.6 2.420743 102.6 1.16619 1101.449138 126.8 2.332381

See FIGS. 4 and 5 Hematocrit Concentration:

Media SEM Media SEM Media SEM Media SEM 1 M 2 M 3 M 4 M Day 5 (8 hours)54.6 1.029563 54 0.83666 52.4 0.4 44.4 0.6 Day 5 (12 hours) 48.6 0.450.8 0.374166 50.6 0.6 46.2 0.583095 Day 5 (16 hours) 46.6 0.4 49.20.374166 48.4 0.509902 45 0.316228 Day 5 (20 hours) 45 0.547723 48.40.678233 45.2 0.860233 43 0.632456 Day 6 44.4 1.32665 48 1.516575 46.62.249444 44 0.948683 1 H 2 H 3 H 4 H Day 5 (8 hours) 55.6 1.32665 54.80.734847 53 0.547723 46.6 0.4 Day 5 (12 hours) 47.2 0.2 50.4 0.67823350.6 0.509902 47.2 0.583095 Day 5 (16 hours) 45.8 0.2 49.6 0.6 47.8 0.245.4 0.979796 Day 5 (20 hours) 44.4 0.509902 47.8 0.374166 45.8 1.2 44.80.860233 Day 6 43.2 0.374166 45.4 1.630951 41.4 1.860107 42.2 1.772004

See FIGS. 6 and 7

Natremia (mmol/L):

Media SEM Media SEM Media SEM Media SEM 1 M 2 M 3 M 4 M Day 5 (8 hours)200.8 3.624914 194.6 5.1049 193 4.312772 175 1.341641 Day 5 (12 hours)178.4 1.32665 191.2 1.655295 192.4 3.893584 169.4 3.059412 Day 5 (16hours) 175.2 1.655295 190.4 1.363818 192 1.923538 173.4 2.420743 Day 5(20 hours) 175.6 1.50333 187.2 0.7348469 189.2 1.933908 173.2 1.496663Day 6 175.2 1.593738 185.2 1.714643 187 1.48324 174.4 2.088061 1 H 2 H 3H 4 H Day 5 (8 hours) 190.2 4.97393 194.8 3.15278 185.6 1.8868 175.21.06771 Day 5 (12 hours) 173.6 1.46969 192.4 2.01494 180.2 1.06771 172.61.36382 Day 5 (16 hours) 169.8 1.35647 189.4 2.37907 177.2 1.15758 171.23.77359 Day 5 (20 hours) 173.2 1.35647 188.4 1.43527 172 1.09545 174.42.37907 Day 6 171.4 3.58608 185.2 1.88149 171.8 1.98494 174.6 1.20831

See FIGS. 8 and 9

Kalemia (mmol/L):

Media SEM Media SEM Media SEM Media SEM 1 M 2 M 3 M 4 M Day 5 (8 hours)5.06 0.478121 4.87 0.4895406 5.62 0.3527038 8.48 0.2009974 Day 5 (12hours) 6.76 0.552811 5.75 0.2792848 5.64 0.3187475 8.4 0.2167949 Day 5(16 hours) 7.94 0.143527 6.56 0.2466778 6.1 0.1294218 8.139 0.1363817Day 5 (20 hours) 8.16 0.129807 7.33 0.1299999 6.79 0.1372953 8.360.1784657 Day 6 8.23 0.128062 7.5 0.1695582 7.55 0.2720294 8.4 0.09354151 H 2 H 3 H 4 H Day 5 (8 hours) 5.06 0.26429 5.75 0.57619 4.9 0.541068.34 0.14 Day 5 (12 hours) 6.52 0.69401 6.46 0.52617 5.81 0.12288 8.420.08456 Day 5 (16 hours) 8.58 0.09028 7.02 0.157 6.47 0.20224 8.3090.11662 Day 5 (20 hours) 8.38 0.10794 7.41 0.17986 6.88 0.12806 8.510.12787 Day 6 8.42 0.10794 7.51 0.11225 7.19 0.19261 8.43 0.11023

Conclusions

The osmotic diarrhea model was developed as described in the literature,resulting in significant weight reduction and hematocrit increase due todehydration.

During dehydration process due to fecal excretion, significant loss ofextracellular fluid is produced. Sodium concentration in this fluid isabout 30 times higher compared to potassium concentration. During theprocess of fluid loss, significant loss of solutes is also observed,including sodium and potassium ions, responsible for regulating liquidrestitution in the body. However, the percentage of potassium loss ishigher than that corresponding to sodium. In addition to liquidreduction, this makes the initial dehydration condition show plasmasodium concentration values higher than those belonging to animals thatdid not experienced dehydration, and plasma potassium concentrationvalues lower than those of non-dehydrated animals.

The results obtained from the body mass analysis indicated that, as aconsequence of diarrhea induction, all the experimental groups by thetime treatment was initiated had lost about 20% of their body mass.Thereafter, comparative results showed a significant difference betweenweight regain in the group treated with the formulation of Example 6 ofthe present invention and the group treated with Pedialyte®. Both inmale and female rats after 24 hours of treatment, the formulation of thepresent invention induced recovery of average body mass in theexperimental group.

Hematocrit is the percent of the total volume of whole blood that iscomposed of red blood cells. Hematocrit loss during dehydration due tofecal excretion is negligible. This implies that the reduction of plasmaextracellular fluid makes hematocrit increase.

On the basis of the condition at the time treatment was initiated, it ispossible to see that all the induced groups have a hematocrit levelhigher than 50%, where all normal values always range from 40% to 50%.The treatment results indicate that recovery in the hematocrit level inthe group treated with the formulation of the present invention wassignificantly faster than that achieved by Pedialyte®. In male rats,normal level was achieved after 24 hours of treatment, whereas in femalerats the action was much more effective, the normal level beingrecovered after 8 hours of treatment.

Natremia and Kalemia analyses are highly influenced by extracellularfluid recovery. Reduction in sodium concentration in all theexperimental groups does not mean there is cation loss, but a reductionin cation concentration. This means the body absorbs sodium and recoversa higher liquid percentage; thus, its concentration diminishes. Theexperimental results revealed that both in male and female rats, therecovery rate of normal sodium and potassium levels was significantlyhigher for the formulation of the present invention compared toPedialyte®.

It should be noted that the term “osmolality” refers to moles perkilogram and the term “osmolarity” refers to moles per liter. They aredifferent terms, but throughout this description, they could be usedinterchangeably due to low density values that enhance the low impact onthe conversion of osmolality to osmolarity of the liposomal rehydrationsalt formulation as described.

It is also possible to use a modified manufacturing process than thatdescribed above by not creating an ethanol phase, and instead, creatinga concentrated phase that already has the liposomes. That concentratedphase is then mixed as an ingredient with the end product. It has alsobeen found that the inclusion level of salts can be increased to as highas 70% in some conditions versus inclusion levels such as 54% or 56%. Inan aspect of manufacturing, the concentrated phase of liposomes has beendeveloped in a ratio of 1:20 and the end product profile has beenupgraded, replacing HFCS such as high fructose corn syrup, with glucoseand Stevia and other sugars while also using natural flavors and colors.A variety of ranges are identified above for the liposomal rehydrationsalt formulation. A specific composition/formulation specificationexplained below shows the different specifications for sodium,potassium, chloride, citrate, glucose, carbohydrates and calories. Thiscurrent example is also known as Speedlyte® and is later compared toanother non-liposome formulation sold by Abbot as Pedialyte® andcompared to WHO (World Health Organization) 2002 recommendations.

Example Composition/Formulation Specifications

Sodium 1,035 mg/L 45 mEq/L Potassium   782 mg/L 20 mEq/L Chloride 1,380mg/L 39 mEq/L Citrate   748 mg/L 8.7 mEq/L  Glucose 13.50 g/L 75 mEq/LTotal Carbohydrates  25.5 grams Calories 90

In this example, based on liposome electrolytes, the actualcomposition/formulation osmolarity should be taken as 125.8 mmol/L basedon a current theoretical osmolarity of 188, with 54% of the electrolytesbeing encapsulated.

These example values in the table above can vary from 5%, 10%, 15%, 20%and 25% above and below these values, although the greater percentagedifference from the listed values is less desirable. Based on theliposome electrolytes, the actual composition/formulation osmolarityshould be taken as 125.8 mmol/l based on a current theoreticalosmolarity of 188, with 54% of the electrolytes being encapsulated. Theosmolarity analysis (meq/L) is referred to as milliequivalents of soluteper liter of solvent and is the amount of substance that reacts or isequivalent to another amount of substance. Of course, the equivalent isthe amount of a substance needed to react with or supply one or morehydrogen ions in an acid-base reaction or react with or supply one ormore of electrons in a redox reaction.

Osmolarity Analysis (meq/L)

Speedlyte ® WHO 2002 Pedialyte ® (conventional/liposomed) Sodium 75 4545/20.2 Chloride 65 35 39/17.5 Potassium 29 20 20/9   Citrate 10 109/4   Glucose 75 139 75/75   Total 245 249 188/125.8 OsmolarityValues can vary from 5%, 10%, 15%, 20% and 25% for the currentformulation.

In the human body, the amount of a substance and equivalence is a verysmall magnitude and it is routinely described in terms ofmilliequivalents (meq) as the measure having been multiplied by 1,000.The osmolarity analysis comparing the World Health Organization 2002standards (WHO 2002) with the formulation manufactured by Abbot asPedialyte® and an example of the current formulation also referred to asSpeedlyte® are illustrated above and compare the example values. ThePedialyte® composition is a non-liposomal formulation that includes anumber ingredients as an oral fluid and electrolyte replacement. Asshown in the table, it is evident that its osmolarity is much higherthan the osmolarity of the current formulation. One differentiatingfactor in the marketplace and for efficacy is the lower osmolarity ofthe current formulation and also has beneficial aspects in its higherabsorption results. Also, the electrolytes and liposomes serve as eithera maintaining formula or a rehydration formula. The percentage deviationranges described above are applicable and the current formulation asshown in the tables has 45 mEq/L of sodium and 20 mEq/L of potassiumfrom 2.28 g/L of sodium chloride plus 2.04 g/L of potassium citrate and0.5 g/L of sodium citrate. The more important electrolyte is sodium andthat can range from 12 mEq/L to as high as 90 mEq/L. Other intermediateranges have been found acceptable such as 20 to 70, 30 to 60, 35 to 55,and 40 to 50 mEq/L for the sodium. The potassium electrolytes can alsovary based on the percentages described above and in one example isabout 15 mEq/L to 25 mEq/L. Phospholipids help create the liposomes andthe liposome concentrations and the range identified above is 1 to 60g/L with other ranges as defined in the examples with 1 to 30, 1 to 40,or 1 to 50 g/L with the specific concentrations of 30, 40 and 50. Theexample formulation as described in the tables above has 2.5 g/L ofphospholipids and that value can range from about 1 to 5 g/L, 1 to 10g/L, in a preferred example, 5 to 10 g/L and up to 30, 40, 50 or 60 g/L.

The amount and type of carbohydrates may vary and in one example, thecarbohydrates are at a concentration of up to 6 g/L and up to 30 g/L.The current formulation as described in the tables has as carbohydrates13.5 g/L of glucose and 11.5 g/L of sugar, and in an example may rangefrom about 8.0 g/L to about 15.0 g/L of at least one additional sugar.The level of carbohydrates may be increased to as high as 70 g/L toappeal to a more mainstream product and it may be lowered to almost 0g/L to appeal to diabetics and the elderly. Those ranges of glucose andsugar can vary from their mid-range value at 5%, 10%, 15%, 20%, and 25%differences above and below.

The size of the liposomes are important for absorption as discussedabove and can vary as noted above and is preferred about at 225 to 450nm, but can range in an example from 200 to 500 nm. Although someavailable commercial products have smaller sized liposomes as alleged bytheir manufacturers, such as 100 mm, it has been determined their levelof inclusion volume is low. Other oral liposome products, for example,using vitamin C, may have liposome sizes greater than 500 nm.

In the example 7 described above, natural flavors are concentrated atabout 1.5 g/L. Natural flavorings and masking flavors may be at aconcentration of up to 5 g/L. The example formulation shown in thecharts above has about 3.12 g/L flavorings and these values could varyfrom 5%, 10%, 15%, 25%, or 25% above and below those values. One examplerange is about 1.0 g/L to about 3.5 g/L. Stevia may be used as a naturalsweetener and in example, the Stevia in the examples above is indicatedat a concentration of about 0.1 to 0.2 g/L, and in the example shown inthe tables, is currently about 0.15 g/L, but it is possible to go ashigh as 0.22 in one example. One example range is about 1.0 g/L to about3.5 g/L.

The liposomal rehydration salt formulation not only maintains hydration,but also operates to rehydrate those persons that are dehydrated, insome cases serious. For example, it is possible to rehydrate and avoidthe intravenous (IV) fluid delivery necessary in some cases because theformulation may be orally administered to humans suffering fromdehydration caused by various factors as indicated below or suffering amoderate to severe dehydration with the need for IV fluids. Theformulation also will allow fluid maintenance and avoid use of IV fluidsby preventing severe dehydration while maintaining body electrolytes andfluids. The formulation may be used for rehydration due to stomach bugsin children and adults and hydration to prevent hangover or rehydrationwhile in a hangover episode. The liposomal rehydration salt formulationmay prevent or avoid the need for parenteral hydration, corresponding tofluids that are injected subcutaneously such as parenteral glucose orsaline.

The liposomal rehydration salt formulation may be used for hydration orrehydration for pregnant or breast-feeding women and for persons thatneed hydration or rehydration due to exercise (sport), outdooractivities, extreme weather or high-altitude conditions, skin burns,flying, hangover episodes, diarrhea, vomiting, high fever, stomach bugsor other types of gastritis, norovirus, rotavirus, and other types ofbacteria and infections. Hikers that are climbing steep hills or cliffsmay also find the liposomal rehydration salt formulation advantageous tohelp in maintaining body electrolytes and in hydration or rehydration.

The liposomal rehydration salt formulation may also be used forhydration or rehydration to different populations, including patientswith parenteral and enteral nutrition, to reduce the volume andconsequently the time of intravenous (IV) treatments. It may be usedwith celiac patients, particularly during an episode such as when aperson may inadvertently eat protein to which they may be allergic suchas found in wheat, rye, and barley and their body mounts an immuneresponse. The symptoms may include abdominal bloating, pain, gas,diarrhea, pale stools and weight loss. The liposomal rehydration saltformulation may also be used with elderly patients, pediatric patients,pregnant and breast-feeding patients, and diabetic patients,particularly those under a SGLT2 inhibitor treatment corresponding to aclass of prescription medications that inhibit sodium-glucose transportprotein 2 and that react to reduce blood to glucose levels. Thus, theliposomal rehydration salt formulation can be especially effective forthose type of patients. Such class of medications are sometimes referredto as gliflozin drugs that inhibit the reabsorption of glucose in thekidney and therefore lower the blood sugar, sometimes too much.

The liposomal rehydration salt formulation as described may also be usedfor those that suffer from intestinal failure, including the Short BowelSyndrome, also called short gut, as a malabsorption disorder caused bythe lack of a functioning small intestine. Diarrhea is a typical symptomthat sometimes results in dehydration, malnutrition and weight loss.

Other populations that will benefit from the liposomal rehydration saltformulation include those that suffer from Cycling Vomiting Syndrome(CVS) with the sudden, repeated attacks as episodes of severe nausea,vomiting and physical exhaustion that could last a few hours to severaldays. Other persons suffering from gastroparesis as, for example,delayed gastric emptying with paresis of the stomach and food oftenremaining in the stomach for an abnormally long time, which may causechronic nausea and vomiting in some cases with erratic blood glucoselevels. Those suffering from Postural Orthostatic Tachycardia Syndrome(POTS) characterized when too little blood returns to the heart whenmoving from a lying down to a standing up position corresponding toorthostatic intolerance may benefit from the formulation. Thosesuffering from ulcerative colitis and colon cancer could use theliposomal rehydration salt formulation to benefit them since often theybecome dehydrated.

Also, those having dysphagia and difficulty swallowing could benefit aswell as those with Sjogren's syndrome, corresponding to a systemicautoimmune disease that may include dry eyes and dry mouth and often isaccompanied by rheumatoid arthritis and lupus. Those with lupus orsimilar immune disorders may also benefit from the liposomal rehydrationsalt formulation. Especially beneficial would be those suffering fromCrohn's Disease and lupus with typical abdominal cramping and pain aspart of a chronic Inflammatory Bowel Disease (IBD) and inflammation ofthe digestive or gastrointestinal (GI) tract. Crohn's Disease is usuallylimited to the end of the small intestine, as compared to ulcerativecolitis, which is usually limited to the large intestine such as thecolon and rectum. The liposomal rehydration salt formulation isbeneficial for sufferers of either disorder. Those having kidney diseasewith dangerous levels of fluid, electrolytes and waste build up willbenefit from the use of the liposomal rehydration salt formulation.

Those suffering from HIV are especially benefitted since often treatmentfor HIV and AIDS causes vomiting and diarrhea. Diarrhea is a typicalside effect that accompanies use of HIV medications used for AIDStreatment. Often this includes nausea and headache with some fever,accompanied by vomiting and diarrhea. The liposomal rehydration saltformulation is especially beneficial in this type of treatment not onlyto help maintain electrolytes, but for rehydration. Some antiretroviraldrugs for AIDS may increase blood sugar and diabetes and the liposomalrehydration salt formulation is beneficial.

Those with the Inflammatory Bowel Disease (IBD) such as Crohn's Diseaseand ulcerative colitis would benefit as well as those with an ostomy,i.e., a stoma as a surgically created opening between the intestines andabdominal wall, and thus typically requiring a bag or pouch. This maycan cause glucose levels and other electrolyte changes in the body,especially in the blood and intestines. Those suffering from microvillusinclusion disease also termed Davidson's Disease also suffer fromchronic, intractable diarrhea causing metabolic acidosis and severedehydration and thus would benefit from use of the liposomal rehydrationsalt formulation. Those suffering from cystic fibrosis (CF) wouldbenefit from its use. Although CF is a genetic order affecting thelungs, it may also affect the pancreas, liver, kidneys and intestinescausing difficulty in breathing. It can also cause fatty stool. Theliposomal rehydration salt formulation can be used in many differenttypes of cancer treatment and especially those suffering from HIVsymptoms.

The liposomal rehydration salt formulation has superior taste, higherabsorption, less intake and lower sugar than other drinks andformulations both using and not using liposomal technology. Testimonieshave stated that individuals feel better hydrated after taking thedisclosed liposomal rehydration salt formulation and have fewerheadaches and less need for IV fluids. Taken regularly, it especiallymay provide those suffering from gastroparesis, Crohn's Disease,ulcerative colitis, POTS, SBS, and colorectal cancer to reduce IVhydration and hospital visits, increase energy, reduce palpitations,feel less thirst, experience fewer headaches, eliminate cramps andreduce dizziness. It is found that the liposomal rehydration saltformulation hydrates in one-third of the time and requires onlyone-third of the intake as compared to many other hydration drinks. Thecurrent formulation also uses 46% less sugar than many commerciallyavailable hydration drinks. The current formulation aids those that livewith the risk of dehydration and it is better than water since water isa very poor hydrator for moderate and severe situations and may causeloss of fluid greater than the amount of fluid consumed, thus leading toelectrolyte imbalance. Even coconut water often may not contain enoughelectrolytes for maintaining proper hydration in severe and chronicdehydration situations. Usually, sports drinks do not contain enoughelectrolytes because they are designed for mild dehydration situations.The electrolyte powders and tablets that are commonly available presentabsorption limitations and bad taste. Many commercially available oralrehydration solutions (ORS) are also old technology as compared to thecurrent liposomal rehydration salt formulation that usesnano-electrolytes for higher fluid absorption, less intake requirementsand better taste. The formulation can be diluted with water, juices andother drinks, depending on the electrolyte level required.

It is possible in some cases to add small amounts of other functionalingredients as part of the liposome technology, including vaccines,drugs, amino acids, mineral salts, vitamins, nutraceuticals, probiotics,prebiotics, and other flavors, including nutritive and non-nutritivesweeteners. The amounts would vary of course depending on end uses.

Many modifications and other embodiments of the invention will come tothe mind of one skilled in the art having the benefit of the teachingspresented in the foregoing descriptions and the associated drawings.Therefore, it is understood that the invention is not to be limited tothe specific embodiments disclosed, and that modifications andembodiments are intended to be included within the scope of the appendedclaims.

That which is claimed is:
 1. A liposomal rehydration salt formulation,comprising phospholipids at a concentration of about 1.0 g/L to 10.0g/L, salts, water, and a percentage inclusion ratio of salts (saltsretained within total salts/liposomes) of at least 50% and a sodiumelectrolyte of about 12 mEq/L to 90 mEq/L, wherein said formulation hasan actual osmolarity lower than 130 based on the at least 50%encapsulation of the salts and said liposomes comprise a particlediameter ranging from 200 nm to 500 nm.
 2. The liposomal rehydrationsalt formulation of claim 1, wherein said sodium electrolyte is fromabout 35 mEq/L to 55 mEq/L.
 3. The liposomal rehydration saltformulation of claim 1, further comprising about 15 mEq/L to 25 mEq/L ofpotassium electrolyte.
 4. The liposomal rehydration salt formulation ofclaim 1, wherein said phospholipids are selected from the groupconsisting of phosphatidylcholines (PCs), phosphatidylserines (PSs),phosphatidylethanolamines (PEs), phosphatidylglycerols (PGs),phosphatidylinositols (PIs), phosphatidic acids (PAs), and mixturesthereof.
 5. The liposomal rehydration salt formulation according toclaim 1, wherein said formulation further comprises an antioxidantselected from the group consisting of phytosterol, tocopherol, andmixtures thereof.
 6. The liposomal rehydration salt formulation of claim1, wherein said salts are selected from the group consisting of sodiumchloride at a concentration of 0.7 g/L to 2.8 g/L, potassium citrate ata concentration of 0.8 g/L to 2.5 g/L, sodium citrate at a concentrationof 0.5 g/L to 2.9 g/L, and mixtures thereof.
 7. The liposomalrehydration salt formulation of claim 1, wherein said formulationfurther comprises about 10 g/L to 17 g/L of glucose and about 8.0 g/L to15 g/L of at least one additional sugar.
 8. The liposomal rehydrationsalt formulation of claim 1, wherein said formulation further comprisesStevia at a concentration of about 0.1 g/L to 0.25 g/L.
 9. The liposomalrehydration salt formulation of claim 1, wherein said formulationfurther comprises natural flavours at a concentration of about 1 g/L to3.5 g/L.
 10. A method of preventing severe dehydration and maintainingbody electrolytes and fluids in a human, comprising orally administeringa liposomal rehydration salt formulation comprising phospholipids at aconcentration of about 1.0 g/L to 10.0 g/L, salts, water, and apercentage inclusion ratio of salts (salts retained within totalsalts/liposomes) of at least 50% and a sodium electrolyte of about 12mEq/L to 90 mEq/L, wherein the formulation has an actual osmolaritylower than 130 based on the at least 50% encapsulation of the salts andthe liposomes comprise a particle diameter ranging from 200 nm to 500nm.
 11. The method according to claim 10, wherein the liposomalrehydration salt formulation is formulated for oral administration foruse by humans that are pregnant or breast-feeding or engaged in one ormore of sport exercises, outdoor activities, extreme weather activities,climbing and flying.
 12. The method according to claim 10, wherein theliposomal rehydration salt formulation is formulated for oraladministration for use by patients having one or more of stomachailments, skin burns, parenteral or enteral nutrition ailments, celiacdisorders, diabetes, SGLT2 inhibitor treatment disorders, intestinalfailure, Short Bowel Syndrome, Cycling Vomiting Syndrome, Gastroparesis,Postural Orthostatic Tachycardia Syndrome, Ulcerative Colitis, ColonCancer, Dysphagia, Sjogren Syndrome, Crohn's disease, Lupus, Alzheimer'sdisease, Renal complications, HIV, Inflammatory Bowel Disease, anOstomy, Microvillus Inclusion Disease, and Cystic Fibrosis.
 13. Themethod of claim 10, wherein said sodium electrolyte is from about 35mEq/L to 55 mEq/L.
 14. The method of claim 10, wherein the liposomalrehydration salt formulation comprises a potassium electrolyte andadministering about 15 mEq/L to 25 mEq/L of the potassium electrolyte.15. The method of claim 10, wherein the liposomal rehydration saltformulation comprises about 10 g/L to 17 g/L of glucose and 8.0 g/L to15 g/L of at least one additional sugar.
 16. A method of rehydrating ahuman suffering from dehydration comprising orally administering aliposomal rehydration salt formulation comprising phospholipids at aconcentration of about 1.0 g/L to 10.0 g/L, salts, water, and apercentage inclusion ratio of salts (salts retained within totalsalts/liposomes) of at least 50% and a sodium electrolyte of about 12mEq/L to 90 mEq/L, wherein the formulation has an actual osmolaritylower than 130 based on the at least 50% encapsulation of the salts andthe liposomes comprise a particle diameter ranging from 200 nm to 500nm.
 17. The method according to claim 16, wherein the liposomalrehydration salt formulation is formulated for oral administration foruse by humans that are pregnant or breast-feeding or engaged in one ormore of sport exercises, outdoor activities, extreme weather activities,climbing and flying.
 18. The method according to claim 16, wherein theliposomal rehydration salt formulation is formulated for oraladministration for use by patients having one or more of stomachailments, skin burns, parenteral or enteral nutrition ailments, celiacdisorders, diabetes, SGLT2 inhibitor treatment disorders, intestinalfailure, Short Bowel Syndrome, Cycling Vomiting Syndrome, Gastroparesis,Postural Orthostatic Tachycardia Syndrome, Ulcerative Colitis, ColonCancer, Dysphagia, Sjogren Syndrome, Crohn's disease, Lupus, Alzheimer'sdisease, Renal complications, HIV, Inflammatory Bowel Disease, anOstomy, Microvillus Inclusion Disease, and Cystic Fibrosis.
 19. Themethod of claim 16, wherein said sodium electrolyte is from about 35mEq/L to 55 mEq/L.
 20. The method of claim 16, wherein the liposomalrehydration salt formulation comprises a potassium electrolyte andadministering about 15 mEq/L to 25 mEq/L of the potassium electrolyte.21. The method of claim 16, wherein the liposomal rehydration saltformulation comprises about 10 g/L to 17 g/L of glucose and 8.0 g/L to15 g/L of at least one additional sugar.